Wine contain L-malic acid in the range of 1 to 10 g/L. The amount of malic acid depends largely on the climatic conditions. Hence, wines produced in colder areas tend to have a relative higher concentration. From a taste and flavour point of view, malic acid is considered undesirable in most red wines and in several types of white wines and sparkling wines and from a practically/economically point of view malic acid is undesirable as it can support growth of bacteria in the wine after it has been bottled.
During the alcoholic fermentation only a small part of the present malic acid is degraded by the yeast. However, the content of malic acid in a wine may be effectively reduced by the so-called malolactic fermentation (MLF) which normally occurs after completion of the alcoholic fermentation. The MLF results from the catabolic activity of various lactic acid bacteria, including species belonging to the genera of Lactobaccilus, Pediococcus and Oenococcus. 
The bacteria may be present in the wine as part of the indigenous microbial flora, or they may have been added as a bacterial starter culture. The most preferable bacteria species by winemakers is Oenococcus oeni, formerly known as Leuconostoc oenos. The catabolic phase is usually entered when the malolactically active bacteria during the growth phase has reached a population density of about 106 colony forming units (CFU) per ml in the wine. During the MLF the bacteria degrade the malic acid, a dicarboxylic acid, to lactic acid, a monocarboxylic acid. As a result of this the acidity of the wine decreases and the pH increases, resulting in a wine with softer palate. Within the field the malolactic fermentation is considered completed when the concentration of malic acid in the wine is less than 30 mg/l (Zoecklein et al., in Wine Analysis and Production, Chapman and Hall, p 296).
However, during the malolactic fermentation Oenococcus oeni also degrade the citric acid, which normally is present in the wine at a concentration of 0.3-0.9 g/L. The catabolism of malic acid and citric acid is in general not concomitant, but sequential, i.e. the citric acid degradation is delayed compared to the malic acid fermentation (see for example Nielsen et al., 1999, Appl. Environ. Microbiol., 65:740-745 and Viljakainen and Laakso, 2000, Eur. Food Res. Technol., 211:438-442). The degree of delay is dependent on the specific bacteria and the wine. The degradation of citric acid is often undesired as the wine looses some of its fruitiness which is appreciated in most wines. Another undesired and unavoidable effect of the citric acid degradation by Oenococcus oeni during MLF is the production of acetic acid, which is one of the end products from the citric acid degradation. The acetic acid concentration in the wine may increase with 0.1-0.3 g/L during the MLF. Acetic acid is highly undesired as it gives the wine an unpleasant vinegar flavour at higher concentration and the winemaker use much attention to keep the acetic acid concentration in the wine as low as possible during the different winemaking steps. Besides the lactic acid bacteria the other sources of acetic acid in winemaking is yeast and acetic acid bacteria. An important intermediary compound in the metabolism of citric acid by Oenococcus oeni is diacetyl. When present at concentration above the sensory threshold, diacetyl gives the wine a buttery aroma which is undesired in most red wines. However, in some white wines, e.g. Chardonnay the aroma may be appreciated.
Because the winemaker often prefer to exercise a greater degree of control over the malolactic fermentation process it has become increasingly common practice in the wine industry to induce the fermentation by use of commercially available concentrated frozen or freeze dried starter cultures of Oenococcus oeni, which are inoculated directly into the wine without any prior re-hydration and activation steps. The usefulness of a concentrated frozen or freeze dried culture which are inoculated direct into wine depends very much on the survival rate of the bacteria culture after the inoculation. The transition of the bacteria from a friendly propagation medium with high pH and high nutrition level to the hostile condition in wine with high ethanol concentration, low pH, low nutrition level, and presence of SO2 is very stressful for the bacteria and may result in survival rates below 1% in the inoculated wine.
Typically, the described propagation medium for lactic acid bacteria, including Oenococcus oeni, contain various nutrients such as vitamins, minerals, yeast extract and sugars. The amount of sugars in the media are normally within 20-50 g/L as higher concentrations of sugars are considered to strongly reduce or inhibit the growth of lactic acid bacteria. In the prior art higher concentrations of sugars in the propagation medium have therefore been considered to be of no practically use or economically interest.
Freeze dried cultures of a strain of Oenococcus oeni, which do not ferment the citric acid in the wine during the malolactic fermentation, are commercially available. There are several advantages in using this strain for malolactic fermentation in wine. The fruitiness of the wine is conserved, there is no production of acetic acid during the malolactic fermentation and there is no production of the buttery flavour, diacetyl. However, the commercial cultures of this strain on the market can not be used for direct inoculation into wine as this results in a survival rate which is of no practical use. According to the commercial application manuals of the products the freeze dried cultures needs re-hydration and an adaptation procedure involving several steps before the bacteria can be inoculated into wine. The first step may involve dissolving the freeze dried product in 5 L of half water, half grape juice or wine added different nutrition. After 5-7 days incubation the pre-culture is diluted with 20-200 L wine which is incubated for another 2-12 days before the final inoculation into the production tank with wine. The adaptation steps are very time and labour consuming for the winemaker as he has to follow the adaptation steps carefully, and there is a high risk for contamination with spoilage bacteria and yeast as the adaptation steps under the practical conditions in the wineries can not be conducted under sterile conditions.
WO 9320180 discloses a method for inducing malolactic fermentation in wine or fruit juice by the direct inoculation of a concentrate of a starter culture containing a selected malolactically active bacteria strain having a survival rate of at least 80% when introduced into a wine having a pH of 3.2 or lower and containing at least 25 mg SO2 per L and at least 12 vol % ethanol, and capable of starting malolactic fermentation when added directly to the wine or fruit juice at a concentration of less than 107 colony forming units per ml. Said bacteria strain is also capable of degrading citric acid. The document clearly indicates that the bacteria strains ability to survive the direct inoculation into wine is linked to the genetic of the strain selected. According to the disclosure the selected bacteria strains are propagated according to processes which are well-known in the art.
U.S. Pat. No. 6,284,518 B1 discloses a media for propagation of different lactic acid bacteria species, including Oenococcus oeni. According to the disclosure a key feature of the invention is the use of a fructose/glucose mixture where fructose is the primary carbohydrate source. Fructose/glucose mixtures containing between 3% to about 45% glucose can be employed, preferably the amount is between 5% and about 40% glucose. Most preferable, the amount is less then 20% of the mixture. In the disclosed media, as biomass growth occurs, the pH is allowed to drop. The amount of carbohydrate source in the disclosed medium is between 30 g/L to 60 g/L. They document describes that 106-107 bacteria/ml are inoculated into the medium and the bacteria are then propagated for 6-8 days before the bacteria can be inoculated into wine.
In U.S. Pat. No. 4,562,077 is disclosed a method for the reduction of malic acid to lactic acid in wine which comprises the inoculation of a culture concentrate into non-sterile fruit juice containing a nitrogen source to provide about 107 to 1010 bacteria per ml. After this activation step the mixture of fruit juice and bacteria are introduced into wine or grape must to produce wine having a reduced malic acid content. The activation conditions disclosed are an activation period of 48 to 72 hours at 18° C. to 25° C.
Carrié et al., 2002, Revue des OEnologues 103: 16-18 describes various malic acid fermenting bacteria, some of which can be inoculated directly into wine. The document furthermore describes the citric acid concentration in wine after/during malolactic fermentation with these bacteria. It appears from FIG. 1 of the document that malolactic fermentation is completed for only one of the strains investigated (strain F). Thus, citric acid concentration is determined prior to or at completion of malolactic fermentation. Because citric acid fermentation is delayed compared to malic acid fermentation (see herein above), citric acid concentration is determined prior to completion of Citric acid degradation. The document is silent regarding whether the bacteria disclosed therein are capable of degrading citric acid. Because lactic acid bacteria normally are capable of degrading citric acid, it must be anticipated that the bacteria disclosed in the document are capable of degrading citric acid.
Nielsen et al., 1996 Am. J. Enol. Vitic. 47:42-48 describes Leuconostoc oenos bacterial cultures capable of fermenting malic acid to lactic acid. The bacteria can be inoculated directly into wine. The bacteria are capable of degrading citric acid.
Nielsen and Richelieu, 1999, Appl. Environ. Microbiol. 65:740-745 describes malolactic fermention and describes that citric acid degradation is delayed compared to degradation of malic acid.
U.S. Pat. No. 5,460,837 describes a method of initiating malolactic fermentation by cultivating malolactically active bacteria in a medium comprising 0.05 to 0.5% glucose, separation of biomass and optionally freeze drying. The freeze dried cultures may be inoculated directly in wine.